3D printed TCP-based scaffold incorporating VEGF-loaded PLGA microspheres for craniofacial tissue engineering.

OBJECTIVE: Vascularization is a critical process during bone regeneration/repair and the lack of tissue vascularization is recognized as a major challenge in applying bone tissue engineering methods for cranial and maxillofacial surgeries. The aim of our study is to fabricate a vascular endothelial growth factor (VEGF)-loaded gelatin/alginate/ß-TCP composite scaffold by 3D printing method using a computer-assisted design (CAD) model. METHODS: The paste, composed of (VEGF-loaded PLGA)-containing gelatin/alginate/ß-TCP in water, was loaded into standard Nordson cartridges and promptly employed for printing the scaffolds. Rheological characterization of various gelatin/alginate/ß-TCP formulations led to an optimized paste as a printable bioink at room temperature. RESULTS: The in vitro release kinetics of the loaded VEGF revealed that the designed scaffolds fulfill the bioavailability of VEGF required for vascularization in the early stages of tissue regeneration. The results were confirmed by two times increment of proliferation of human umbilical vein endothelial cells (HUVECs) seeded on the scaffolds after 10 days. The compressive modulus of the scaffolds, 98±11MPa, was found to be in the range of cancellous bone suggesting their potential application for craniofacial tissue engineering. Osteoblast culture on the scaffolds showed that the construct supports cell viability, adhesion and proliferation. It was found that the ALP activity increased over 50% using VEGF-loaded scaffolds after 2 weeks of culture. SIGNIFICANCE: The 3D printed gelatin/alginate/ß-TCP scaffold with slow releasing of VEGF can be considered as a potential candidate for regeneration of craniofacial defects.

Mechanisms of chlorpyrifos and diazinon induced neurotoxicity in cortical culture.

The main action of organophosphorous insecticides is generally believed to be the inhibition of acetylcholinesterase (AChE). However, these compounds also inhibit many other enzymes, any of which may play a role in their toxicity. We tested the neurotoxic mechanism of two organophosphorous insecticides, chlorpyrifos and diazinon in primary cortical cultures. Exposure to the insecticides caused a concentration-dependent toxicity that could not be directly attributed to the oxon forms of the compounds which caused little toxicity but strongly inhibited AChE. Addition of 1 mM acetylcholine or carbachol actually attenuated the toxicity of chlorpyrifos and diazinon, and the muscarinic receptor antagonist, atropine, and the nicotinic receptor antagonist, mecamylamine, did not attenuate the toxicity of either insecticide. These results strongly suggest that the organophosphorous toxicity observed in this culture system is not mediated by buildup of extracellular acetylcholine resulting from inhibition of AChE. The toxicity of chlorpyrifos was attenuated by antagonists of either the NMDA or AMPA/kainate-type glutamate receptors, but the cell death was potentiated by the caspase inhibitor ZVAD. Diazinon toxicity was not affected by glutamate receptor antagonists, but was attenuated by ZVAD. Chlorpyrifos induced diffuse nuclear staining characteristic of necrosis, while diazinon induced chromatin condensation characteristic of apoptosis. Also, chlorpyrifos exposure increased the levels of extracellular glutamate, while diazinon did not. The results suggest two different mechanisms of neurotoxicity of the insecticides, neither one of which involved acetylcholine. Chlorpyrifos induced a glutamate-mediated excitotoxicity, while diazinon induced apoptotic neuronal death.

Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are critical for synaptic plasticity that underlies learning and memory. But, they have also been described as a common source of neuronal damage during stroke and neurodegenerative diseases. Several studies have suggested that cellular location of NMDARs (synaptic or extrasynaptic) is a key parameter controlling their effect on neuronal viability. The aim of the study was to understand the relation between these two pools of receptors and to determine their implication in both beneficial and/or deleterious events related to NMDAR activation. We demonstrated that selective extrasynaptic NMDAR activation, as well as NMDA bath application, does not activate extracellular signal-regulated kinase (ERK) pathways, but induces mitochondrial membrane potential breakdown and triggers cell body and dendrite damages, whereas synaptic NMDAR activation is innocuous and induces a sustained ERK activation. The functional dichotomy between these two NMDAR pools is tightly controlled by glutamate uptake systems. Finally, we demonstrated that the only clinically approved NMDAR antagonist, memantine, preferentially antagonizes extrasynaptic NMDARs. Together, these results suggest that extrasynaptic NMDAR activation contributes to excitotoxicity and that a selective targeting of the extrasynaptic NMDARs represents a promising therapeutic strategy for brain injuries.

Neurotoxicity of dental amalgam is mediated by zinc.

The use of dental amalgam is controversial largely because it contains mercury. We tested whether amalgam caused toxicity in neuronal cultures and whether that toxicity was caused by mercury. In this study, we used cortical cell cultures to show for the first time that amalgam causes nerve cell toxicity in culture. However, the toxicity was not blocked by the mercury chelator, 2,3-dimercaptopropane-1-sulphonate (DMPS), but was blocked by the metal chelator, calcium disodium ethylenediaminetetraacetate (CaEDTA). DMPS was an effective mercury chelator in this system, since it blocked mercury toxicity. Of the components that comprise amalgam (mercury, zinc, tin, copper, and silver), only zinc neurotoxicity was blocked by CaEDTA. These results indicate that amalgam is toxic to nerve cells in culture by releasing zinc. While zinc is known to be neurotoxic, ingestion of zinc is not a major concern because zinc levels in the body are tightly regulated.

Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release.

Central neurons exposed to several types of sublethal stress, including ischemia, acquire resistance to injury induced by subsequent ischemic insults, a phenomenon called ischemic preconditioning. We modeled this phenomenon in vitro, utilizing exposure to 45 mM KCl to reduce the vulnerability of cultured murine cortical neurons to subsequent oxygen-glucose deprivation. Twenty-four hours after preconditioning, cultures exhibited enhanced depolarization-induced, tetanus toxin-sensitive GABA release and a modest decrease in glutamate release. Total cellular GABA levels were unaltered. Inhibition of GABA degradation with the GABA transaminase inhibitor (+/-)-gamma-vinyl GABA, or addition of low levels of GABA, muscimol, or chlormethiazole to the bathing medium, mimicked the neuroprotective effect of preconditioning against oxygen-glucose deprivation-induced death. However, neuronal death was enhanced by higher levels of these manipulations, as well as by prior selective destruction of GABAergic neurons by kainate. Finally, selective blockade of GABA(A) receptors during oxygen-glucose deprivation or removal of GABAergic neurons eliminated the neuroprotective effects of prior preconditioning. Taken together, these data predict that presynaptic alterations, specifically enhanced GABA release together with reduced glutamate release, may be important mediators of ischemic preconditioning, but suggest caution in regard to interventions aimed at increasing GABA(A) receptor activation.

Calcitonin potentiates oxygen-glucose deprivation-induced neuronal death.

Calcitonin is a hormone that decreases plasma calcium through inhibition of osteolysis. It is used in the treatment of osteoporosis and other bone disorders. Salmon calcitonin is typically utilized in individuals for whom use of estrogen is contraindicated, for example, women at high risk for breast cancer. In addition to actions on bone, calcitonin may have effects on the central nervous system. Receptors for calcitonin are present on central neurons, and salmon calcitonin has been shown to alter neuronal activity. Since salmon calcitonin is used clinically, and it can have actions on neurons, the present studies were designed to determine whether salmon calcitonin could alter death of cortical neurons. The effects of salmon calcitonin on neuronal death induced by exposure of murine cortical cultures to serum deprivation, staurosporine, oxygen-glucose deprivation, kainate, and NMDA were tested. Salmon calcitonin had no effect on apoptotic cell death in cortical cultures. However, acute treatment with salmon calcitonin (1-1000 nM) caused significant potentiation of neuronal death induced by oxygen-glucose deprivation. Similarly, salmon calcitonin potentiated cell death induced by exposure to kainate. In contrast, it did not potentiate cell death induced by exposure to NMDA. In fact, addition of a high concentration (1000 nM) of salmon calcitonin attenuated NMDA toxicity. These results indicate that calcitonin is not a survival factor for cortical neurons; however, it can alter excitotoxic cell death. The most interesting, and disturbing, effect is the ability of low concentrations of salmon calcitonin to potentiate oxygen-glucose deprivation-induced cell death.

Zinc-induced neuronal death in cortical neurons.

Although Zn2+ is normally stored and released in the brain, excessive exposure to extracellular Zn2+ can be neurotoxic. The purpose of the present study was to determine the type of neuronal cell death, necrosis versus apoptosis, induced by Zn2+ exposure. Addition of 10-50 microM ZnCl2 to the bathing medium of murine neuronal and glial cell cultures induced, over the next 24 hrs., Zn2+-concentration-dependent neuronal death; some glial death also occurred with Zn2+ concentrations above 30 microM. The neuronal death induced by 20 microM Zn2+ was characterized by coarse chromatin condensation, the formation of apoptotic bodies, and internucleosomal DNA fragmentation. It was attenuated in cortical cell cultures prepared from mice null for the bax gene, and by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (ZVAD, 100 microM), but not by the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV, 200 microM ). In contrast, the neuronal death induced by 50 microM Zn2+ was characterized by plasma membrane disruption and random DNA fragmentation; this death was attenuated by D-APV, but exhibited little sensitivity to ZVAD or deletion of bax. These results suggest that Zn2+ can induce cell death with characteristics of either apoptosis or necrosis, depending on the intensity of the Zn2+ exposure.

Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?

Neuronal apoptosis induced in cortical cultures by exposure to serum deprivation, staurosporine, nifedipine, or C2-ceramide was assayed by lactate dehydrogenase (LDH) release or inhibition of 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction. The protective effects of neurotrophin-4, Z-Val-Ala-Asp-fluoromethylketone (ZVAD), and cycloheximide against each insult were also assayed. The level of injury for each insult was similar whether determined by LDH release or inhibition of MTT reduction, but effects of anti-apoptotic agents were assay dependent. ZVAD and cycloheximide protected neurons from nifedipine-induced death, when assayed by LDH release, but not MTT reduction. In contrast, only cycloheximide attenuated C2-ceramide-induced LDH release, while ZVAD and cycloheximide actually enhanced the C2-ceramide induced inhibition of MTT reduction. Counting of trypan blue positive cells provided results consistent with values obtained using the LDH assay. These results indicate that both LDH release and MTT reduction accurately determine apoptotic death of neurons. However, the MTT assay does not always correctly quantify neuroprotective effects, this likely reflects differences in the point of the death pathway that the neuroprotective agents act. Therefore, while the MTT assay is of limited value in assessing the efficacy of neuroprotective strategies, it may provide information regarding whether specific anti-apoptotic agents act up or downstream of mitochondrial dysfunction.

Selective activation of group II mGluRs with LY354740 does not prevent neuronal excitotoxicity.

Recent reports have suggested a role for group II metabotropic glutamate receptors (mGluRs) in the attenuation of excitotoxicity. Here we examined the effects of the recently available group II agonist (+)-2-Aminobicyclo[3.1.0]hexane-2-6-dicarboxylic acid (LY354740) on N-methyl-D-aspartate (NMDA)-induced excitotoxic neuronal death, as well as on hypoxic-ischemic neuronal death both in vitro and in vivo. At concentrations shown to be selective for group II mGluRs expressed in cell lines (0.1-100 nM), LY354740 did not attenuate NMDA-mediated neuronal death in vitro or in vivo. Furthermore, LY354740 did not attenuate oxygen-glucose deprivation-induced neuronal death in vitro or ischemic infarction after transient middle cerebral artery occlusion in rats. In addition, the neuroprotective effect of another group II agonist, (S)-4-carboxy-3-phenylglycine (4C3HPG), which has shown injury attenuating effects both in vitro and in vivo, was not blocked by the group II antagonists (2 S)-alpha-ethylglutamic acid (EGLU), (RS)-alpha-methyl-4-sulphonophenylglycine (MSPG), or the group III antagonist (S)-alpha-methyl-3-carboxyphenylalanine (MCPA), suggesting that this neuroprotection may be mediated by other effects such as upon group I mGluRs.

Neurotrophin-mediated potentiation of neuronal injury.

The neurotrophins are a diverse family of peptides which activate specific tyrosine kinase-linked receptors. Over the past five decades, since the pioneering work of Levi-Montalcini and colleagues, the critical role that neurotrophins play in shaping the developing nervous system has become increasingly established. These molecules, which include the nerve growth factor (NGF)-related peptides, NGF, brain-derived neurotrophic factor (BDNF), NT-4/5 and NT-3, promote differentiation and survival in the developing nervous system, and to a lesser extent in the adult nervous system. As survival-promoting molecules, neurotrophins have been studied as potential neuroprotective agents, and have shown beneficial effects in many model systems. However, a surprising "dark side" to neurotrophin behavior has emerged from some of these studies implying that, under certain pathological conditions, neurotrophins may exacerbate, rather than alleviate, injury. How neurotrophins cause these deleterious consequences is a question which is only beginning to be answered, but initial work supports altered free radical handling or modification of glutamate receptor expression as possible mechanisms underlying these effects. This review will focus on evidence suggesting that neurotrophins may enhance injury under certain circumstances and on the mechanisms behind these injury-promoting aspects.

Antagonists for group I mGluRs attenuate excitotoxic neuronal death in cortical cultures.

Activation of ion channel-linked glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptors, mediates the excitotoxic effects of glutamate upon central neurons. We examined the hypothesis that activation of group I metabotropic glutamate receptors (mGluRs) would increase NMDA receptor-mediated cortical neuronal death. Addition of the selective group I mGluR agonists, dihydroxyphenylglycine (DHPG) or trans-azetidine-2,4-dicarboxylic acid (t-ADA) potentiated NMDA-induced neuronal death, and application of the group I mGluR-selective antagonist, aminoindan-1,5-dicarboxylic acid (AIDA), as well as the non-selective antagonists methyl-4-carboxyphenylglycine (MCPG) or 4-carboxyphenylglycine (4CPG) reduced NMDA- and kainate-induced neuronal death in murine cortical cultures. The pro-excitotoxic effect of group I mGluR activation may be mediated largely by enhancement of glutamate release, as DHPG potentiated high potassium-stimulated glutamate release, and the protective effects of both AIDA and MCPG were abolished when NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors were blocked immediately after toxic NMDA receptor overstimulation. The present data support the possibility that antagonizing group I mGluRs may be a useful strategy for attenuating excitotoxic neuronal death in certain disease states.

Extracellular acidity potentiates AMPA receptor-mediated cortical neuronal death.

The extracellular acidity that accompanies brain hypoxia-ischemia is known to reduce both NMDA and AMPA-kainate receptor-mediated currents and NMDA receptor-mediated neurotoxicity. Although a protective effect of acidic pH on AMPA-kainate receptor-mediated excitotoxicity has been assumed, such has not been demonstrated. Paradoxically, we found that lowering extracellular pH selectively increased AMPA-kainate receptor-mediated neurotoxicity in neocortical cell cultures, despite reducing peak elevations in intracellular free Ca2+. This injury potentiation may, at least in part, be related to a slowed recovery of intracellular Ca2+ homeostasis, observed after AMPA-kainate receptor activation, but not after NMDA receptor activation or exposure to high K+. The ability of acidic pH to selectively augment AMPA-kainate receptor-mediated excitotoxicity may contribute to the prominent role that these receptors play in selective neuronal death after transient global ischemia.

Conditioning heat stress reduces excitotoxic and apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro.

We investigated the effects of sublethal heat stress in murine cortical cell cultures exposed to combined oxygen and glucose deprivation. Pretreatment with sublethal heat stress mildly attenuated the widespread neuronal death induced a day later by 30-60 min of oxygen-glucose deprivation. Heat stress also blunted the increase in extracellular glutamate concentrations induced by the oxygen-glucose deprivation, as well as the neuronal death and 45Ca2+ uptake induced by exogenous addition of NMDA, although no reduction was seen in neuronal death caused by exogenous kainate or in NMDA-induced whole-cell currents. However, arguing against the idea that the neuroprotective effect of heat stress against neuronal death was exclusively due to reduction of excitotoxicity was the finding that heat stress also reduced the neuronal apoptosis induced by oxygen-glucose deprivation in the presence of glutamate antagonists. This antiapoptotic effect was specific in that heat stress did not reduce neuronal vulnerability to staurosporine-induced apoptosis. Whereas heat stress transiently suppressed protein synthesis, achieving comparable protein synthesis inhibition with cycloheximide did not reproduce the neuroprotective effects of heat stress. These studies suggest that a conditioning heat stress is able to attenuate both the excitotoxic and the apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro, by mechanisms independent of protein synthesis reduction.

Carboxyfullerenes as neuroprotective agents.

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

GABAA receptor activation attenuates excitotoxicity but exacerbates oxygen-glucose deprivation-induced neuronal injury in vitro.

We examined the effects of GABA receptor stimulation on the neuronal death induced by exogenously added excitatory amino acids or combined oxygen-glucose deprivation in mouse cortical cell cultures. Death induced by exposure to NMDA, AMPA, or kainate was attenuated by addition of GABA or the GABAA receptor agonist, muscimol, but not by the GABAB receptor agonist, baclofen. The antiexcitotoxic effect of GABAA receptor agonists was blocked by bicuculline or picrotoxin. In contrast, GABA or muscimol, but not baclofen, markedly increased the neuronal death induced by oxygen-glucose deprivation. Muscimol potentiation of neuronal death was associated with increased glutamate efflux to the bathing medium, and increased cellular 45Ca2+ accumulation; it was blocked by MK-801, but not NBQX, suggesting mediation by NMDA receptors. Bicuculline only weakly attenuated muscimol potentiation of oxygen-glucose deprivation-induced neuronal death, probably because it itself increased this death. Present results raise a note of caution in the proposed use of GABAA receptor stimulation to limit ischemic brain damage in vivo.

Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS.

BACKGROUND AND PURPOSE: Previous studies have shown that brain ischemia and other insults can induce a marked increase in inducible nitric oxide synthase (iNOS) expression in astrocytes and some immune cells, but the biological significance of this phenomenon has not been elucidated. The purpose of the present study was to determine whether this induction of astrocyte iNOS alters neuronal vulnerability to severe hypoxic insults. METHODS: Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1 beta or lipopolysaccharide. Cultures were exposed to combined oxygen-glucose deprivation. The extracellular concentration of glutamate was measured by high-performance liquid chromatography. N-Methyl-D-aspartate (NMDA) receptor activity was assessed by measurement of 45Ca2+ influx: neuronal death was assessed by morphological examination and quantitated by measurement of lactate dehydrogenase efflux to the bathing medium. RESULTS: In murine neocortical cell cultures containing neurons and astrocytes, neuronal injury induced by combined oxygen-glucose deprivation was not reduced by the addition of the nitric oxide synthase inhibitors NG-nitro-L-arginine or LG-nitro-arginine methyl ester. However, after induction of astrocyte iNOS activity with interferon gamma plus lipopolysaccharide or interleukin-1 beta, oxygen-glucose deprivation-induced neuronal injury was markedly enhanced and nitric oxide synthase inhibitors became protective. This iNOS-mediated potentiation was associated with a large increase in both extracellular glutamate accumulation and 45Ca2+ influx into neurons. The potentiation could be blocked by MK-801 but not CNQX, suggesting critical involvement of NMDA receptor activation. CONCLUSIONS: These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor-mediated neuronal death consequent to hypoxic-ischemic insults.

Preincubation with protein synthesis inhibitors protects cortical neurons against oxygen-glucose deprivation-induced death.

Twenty-four hour exposure to cycloheximide produced a concentration-dependent reduction in protein synthesis in mouse cortical cell cultures. Unexpectedly, a 24 h pretreatment with cycloheximide exposure also reduced neuronal vulnerability to subsequent oxygen-glucose deprivation-induced injury, measured both acutely (cell swelling) or after one day (cell lysis). This neuroprotective effect was attenuated if the period of cycloheximide pretreatment was shortened to 8 h, and lost if the pretreatment was shortened to 1 h. A comparable neuroprotective effect was also induced by 24 h pretreatment with another protein synthesis inhibitor, emetine. The neuroprotection induced by pretreatment with cycloheximide or emetine was probably not attributable to reduction of apoptosis: (i) neuronal death under these conditions occurs by N-methyl-D-aspartate receptor-mediated excitotoxic necrosis, not apoptosis; (ii) the same cycloheximide pretreatment did not block staurosporine-induced apoptosis. Also unlikely as an explanation is reduction in postsynaptic vulnerability to excitotoxicity, as death induced by exogenous addition of N-methyl-D-aspartate, kainate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was little affected by cycloheximide pretreatment. Rather, the protective effect of cycloheximide pretreatment was probably explained, at least in part, by marked reduction in the glutamate release induced by oxygen-glucose deprivation.

BDNF or IGF-I potentiates free radical-mediated injury in cortical cell cultures.

Free radical-mediated damage to cultured cortical neurons was induced by a 24 h exposure to Fe2+ (30 microM) or an inhibitor of gamma-glutamylcysteine synthetase, L-buthionine-[S,R]-sulfoximine (BSO, 1 mM). As expected, neuronal death was blocked by inclusion of the free radical scavenger trolox during the Fe2+ or BSO exposure. However, unexpectedly, pretreatment of the cultures with BDNF or IGF-I markedly potentiated neuronal death. This growth factor-potentiated death was still blocked by trolox, but was insensitive to glutamate antagonists. Concurrent addition of cycloheximide with the growth factors prevented injury potentiation. Present findings suggest that growth factors may increase free radical-induced neuronal death by mechanisms dependent upon protein synthesis.

Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro.

Mouse cortical cell cultures exposed to transient oxygen-glucose deprivation developed marked acute cell body swelling followed by neurodegeneration, consistent with necrosis-type death. This death was not attenuated by the protein synthesis inhibitor, cycloheximide, but was attenuated by addition of the N-methyl-D-asparate antagonist, MK-801 (dizocilpine maleate), and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. If the deprivation insult was extended to overcome the protective effect of glutamate antagonists, neuronal death resulted that was associated with cell body shrinkage and DNA fragmentation, and was attenuated by cycloheximide. These data suggest that oxygen-glucose deprivation can induce in cortical neurons both excitotoxic necrosis, and apoptosis dependent on new macromolecule synthesis.

Profound systemic hypothermia inhibits the release of neurotransmitter amino acids in spinal cord ischemia.

Profound hypothermia induced with cardiopulmonary bypass has a protective effect on spinal cord function during operations on the thoracoabdominal aorta. The mechanism of this protection remains unknown. It has been proposed that the release of excitatory amino acids in the extracellular space plays a causal role in irreversible neuronal damage. We investigated the changes in extracellular neurotransmitter amino acid concentrations with the use of in vivo microdialysis in a swine model of spinal cord ischemia. All animals underwent left thoracotomy and right atrium-femoral artery cardiopulmonary bypass with additional aortic arch perfusion. Lumbar laminectomies were then done and microdialysis probes were inserted stereotactically in the anterior horn of the second and fourth segments of the lumbar spinal cord. The probes were perfused with artificial cerebrospinal fluid at a rate of 2 microliters/min and 15-minute samples were assayed by high-performance liquid chromatography. Group 1 animals (n = 6) underwent aortic clamping distal to the left subclavian artery and proximal to the renal arteries for 60 minutes at normothermia (37 degrees C) and group 2 animals (n = 5) were cooled to a rectal temperature of 20 degrees C before application of aortic clamps, maintained at this level during cardiopulmonary bypass until the aorta was unclamped, and then slowly rewarmed to 37 degrees C. Seven amino acids were studied, including two excitatory neurotransmitters (glutamate and aspartate) and five putative inhibitory neurotransmitters (glycine, gamma-aminobutyric acid, serine, adenosine, and taurine). Glutamate exhibited a threefold increase in extracellular concentration during normothermic ischemia compared with baseline values and remained elevated until 60 minutes after reperfusion. The increase in aspartate concentration was not significant. The extracellular concentrations of glycine and gamma-aminobutyric acid also increased significantly during ischemia and reperfusion. Hypothermia uniformly prevented the release of amino acids in the extracellular space. Glutamate levels remained significantly decreased even after rewarming to normothermia whereas glycine levels returned to baseline values. These results are consistent with a role for excitatory amino acids in the production of ischemic spinal cord injury and suggest that the mechanism of hypothermic protection may be related to decreased release of these amino acids in the ischemic spinal cord.